Vitamin D can modulate immune responses, and its deficiency is linked to increased autoimmunity and susceptibility to infection. In the general population, it has been observed that serum vitamin D levels are connected with the risk of COVID-19 and its severity. Our study aims to examine reported findings on the effect of vitamin D serum levels on infection of COVID-19 during pregnancy. PubMed, Web of Science, Embase, and Cochrane Library were searched for relevant studies. Serum vitamin D serum levels in COVID-19-positive and COVID-19-negative pregnant women were 24.61 ± 20.86 ng/mL and 24.12 ± 17.33 ng/mL, respectively. In mild vs. moderate to critical COVID-19 pregnant women, vitamin D serum levels were 16.71 ± 9.04 ng/mL vs. 10.7 ± 9.37 ng/mL and severe vs. non-severe were 13.21 ± 11.47 ng/mL vs. 15.76 ± 10.0 ng/mL. Only one study reported vitamin D serum levels in the placenta of COVID-19-positive pregnant women compared with the control and results varied and amounted to 14.06 ± 0.51 ng/mL vs. 12.45 ± 0.58 ng/mL, respectively. Vitamin D deficiency tends to be common in pregnant women who have COVID-19, and the level of this vitamin has been demonstrated to have a strong correlation with the severity of the illness. As vitamin D serum levels correlate with COVID-19 symptoms and even with its occurrence, appropriate vitamin D supplementation in the prenatal period is suggested. The global impact of the COVID-19 pandemic has been far-reaching, affecting a significant portion of the global population, and almost 6.9 million people have died because of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,4,5. PreCOVID-19 significantly affects the human immune system, reaching beyond the infection of pneumocytes to involve immunological cells. This impact is mediated through the renin–angiotensin system (RAS), where the invasion of cells occurs when SARS-CoV-2 spike proteins bind to angiotensin-converting enzyme 2 (ACE-2) receptors located on cell membranes. This action allows the virus to replicate and invade cells, leading to a reduction in the concentration of ACE-2 receptors and disruption of cellular homeostasis. This process is crucial for the further reproduction and spreading of the virus. Additionally, ACE-2 also plays a vital role in protecting against lung injury, emphasizing the impact of its activity on the severity of pulmonary symptoms observed in COVID-19-infected individuals ,12,13. FTo regulate the immune response, vitamin D promotes the proliferation of Th2 lymphocytes while suppressing the proliferation of Th1 lymphocytes . FurtherEnsuring appropriate lifestyle choices and adequate vitamin supplementation during pregnancy are crucial for optimal fetal development. Among the various vitamin deficiencies, vitamin D insufficiency is particularly common, affecting not only high-risk groups in North American and European countries but also populations in the Middle East and Asia due to modern lifestyles ,28. WideInsufficient levels of vitamin D in the bloodstream contribute to cytokine storms, inadequate protection against apoptosis of epithelial cells, and impaired repair mechanisms of epithelial cells. Consequently, these factors expose the lungs to heightened immune dysregulation, which can have fatal consequences. Moreover, lower serum levels of vitamin D are inversely associated with the presence of pro-inflammatory cytokines, including interleukin 1 (IL-1), interleukin 6 (IL-6), and elevated levels of c-reactive protein (CRP). These cytokines have been linked to the severity of COVID-19 and its adverse outcomes ,42. A stGiven the significance of the presented findings, it is crucial to investigate the impact of vitamin D serum levels on pregnant women affected by COVID-19. This meta-analysis aims to systematically evaluate and analyze previously reported studies examining the relationship between vitamin D serum levels and the risk of infection and the severity of COVID-19 illness during pregnancy.This meta-analysis adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) criteria, and the checklist confirming compliance is provided in the Two independent reviewers (M.P. and L.S.) assessed potentially eligible publications individually, with any dispute resolved through additional discussion or arbitrated by a third reviewer (S.F.). The search was conducted across multiple databases from January 2020 to March 2023, including PubMed, Web of Science, Embase, and the Cochrane Library. Additionally, a search was performed on Google Scholar as a supplementary electronic database. The combination of the following keywords was used: “vitamin D” OR “cholecalciferol” OR “vitamin D3” AND “pregnancy” OR “pregnant” AND “COVID-19” OR “SARS-CoV-2” OR “severe acute respiratory syndrome coronavirus-2”. The reference lists of the included studies were manually reviewed to identify any potentially relevant papers. To avoid duplication, only the most recent or complete reports from the same authors were included. Furthermore, the reference lists of relevant publications and systematic reviews were examined to identify additional potentially relevant studies. All references were imported into Endnote (version X9), and duplicate entries were removed before using Rayyan, a software screening tool .Eligible studies were required to meet the following inclusion criteria: a study comparing vitamin D serum levels in pregnant women with COVID-19 to a control group, mild vs. moderate to critical pregnant women, or severe vs. non-severe pregnant women. Studies that met the following exclusion criteria were excluded: (1) research that failed to produce any of the aforementioned outcomes; (2) studies with no comparable group; (3) studies not published in English; and (4) article types such as editorials, conference papers, reviews, and letters to the editor.Two independent reviewers (M.P. and S.F.) conducted data extraction using a predefined data extraction form prepared by L.S. Any discrepancies or disagreements between the reviewers were resolved through consultations with the third reviewer (L.S.). The extracted information from the eligible publications included the following: study characteristics  and data related to pregnant women . To assess the methodological quality of the included studies, the Newcastle–Ottawa Quality Scale (NOS) was employed. The NOS evaluates the quality of a study based on three criteria: selection, comparability, and exposure. Each criterion can be awarded a maximum of four, two, and three stars, respectively. Studies with NOS scores of 7 or higher were considered to be of good quality .p < 0.05. We approximated the means and standard deviations by using the method that was presented by Hozo et al., where continuous outcomes were reported as a median, range, and interquartile range in the included studies  in the serum, ranging from 30 to 60 ng/mL (75 to 150 nmol/L), has been associated with a reduced risk of severe COVID-19 complications and potential prevention of infection ,77,78. IVitamin D supplementation could decrease C-reactive protein (CRP) levels and influence leukocyte distribution . EntrenaSzarpak et al. conducted a study highlighting the potential reduction in COVID-19 transmission through the implementation of healthy nutritional practices and vitamin D supplementation among vulnerable populations, including pregnant women . This isOur analysis encountered several limitations. The number of published articles investigating the correlation between vitamin D and the severity of COVID-19 symptoms in pregnant women was relatively small, and the study groups were not extensive. Moreover, the included articles were published between 2021 and 2022, which is a very short observation period, highlighting the need for further follow-up research to provide more substantial evidence. Another limitation stems from the heterogeneity and retrospective nature of the included publications. Moreover, the scientific literature indicates that vitamin D levels may decline as the disease progresses, making it challenging to determine whether the differences in vitamin D levels are causally linked to the disease course or represent a consequence of the deficiency. It is important to note that the timing of blood sample collection for measuring serum levels of 25-hydroxyvitamin D (25(OH)D) can significantly influence outcomes. Despite these limitations, the observed trends suggest a potential correlation between low serum levels of 25(OH)D and severe COVID-19 symptoms. Nevertheless, despite these promising findings, further research investigating the level of vitamin D and its supplementation is needed.There is growing evidence supporting the immune-boosting and anti-inflammatory properties of vitamin D. Pregnant women with COVID-19 often experience vitamin D deficiency, and the level of this vitamin has been shown to have a strong correlation with the severity of the disease. Given the correlation between vitamin D serum levels and COVID-19 symptoms, as well as the occurrence of the infection, it is advisable to consider appropriate vitamin D supplementation during pregnancy.


Opuntia ficus-indica (OFI) fruit was compared with gum arabic (GA) as wall materials for the microencapsulation of Colombian blueberry anthocyanins, using the spray-drying process. For both types of microencapsulates, the following were determined: anthocyanin content (UV–vis and HPLC/MS-MS), total dietary content (enzymatic–gravimetric method), antioxidant activity (ORAC), color (CIELab parameters), morphology (SEM and particle size), and thermal behavior (DSC/TGA). Six different anthocyanins were identified by HPLC/MS-MS in the non-lyophilized Andean blueberry sample (LABP) and in the OFI-mucilage and GA microcapsules. OFI mucilage, compared to GA, favors the formation of larger spherical particles, a smoother surface without cracks, and greater thermal stability. The higher anthocyanin retention capacity in OFI microcapsules leads to higher antioxidant capacity and red coloration for this biomaterial. Consequently, the microencapsulation of anthocyanins with mucilage from the peel of the OFI fruit is proposed as a promising alternative for the protection and incorporation of this natural dye with high antioxidant capacity and dietary fiber content in new functional food/cosmetic formulations, while giving added value to the natural byproducts of OFI.The recovery of byproducts from the food industry is a promising approach to obtain hydrophilic biopolymers with potential health benefits. In this work, the mucilage obtained from the peel of the  The incorporation of natural pigments in the formulation of new products in the food, cosmetics, and pharmaceutical industries has become a research area of intense activity and high development. The main objective of this activity is to replace traditional synthetic dyes—such as coal tar derivatives, to which toxic effects are attributed due to the presence of azo groups —with pigVaccinium meridionale Swartz is a wild berry (Andean blueberry) found in the mountains of Colombia, Venezuela, and Ecuador, where it is known as “agraz”. This berry contains high concentrations of anthocyanins (flavonoids)—that is, water-soluble glycosylated anthocyanidins—and is of interest in the food industry due to its desired coloring capacity, as well as for its nutritional and medicinal properties [The fruit of the species operties ,4. Depenoperties . Howeveroperties . The hydoperties .Vaccinium spp.) [Several studies have shown that microencapsulation by spray-drying favors the preservation and bioaccessibility of anthocyanins extracted from blueberries (um spp.) . Selecteum spp.) , whey prum spp.) , whey prum spp.) , maltodeum spp.) , proteinum spp.) , and malum spp.) . HoweverOpuntia ficus-indica fruit peels can be used as a raw biopolymer material for the development of biodegradable packaging.On the other hand, the mucilage extracted from cactaceous plants has shown outstanding ability as a functional encapsulant biomaterial because of its gelling and thickening properties, and its high content of dietary fiber . MucilagOpuntia ficus-indica fruit as a wall material for the spray-drying microencapsulation of anthocyanins from the Colombian Andean blueberry. For comparative purposes, the same anthocyanin microencapsulation procedure was also carried out using gum arabic as the traditional wall material. For both microcapsule formulations, the microstructural (SEM and particle size) and thermal (DSC/TGA) properties were determined, as well as the relative percentages and identification of anthocyanins, color parameters, dietary fiber, and antioxidant activity. For some studies, a comparison with the lyophilized fruit pulp without microencapsulation was also performed.This study investigated the applicability of the mucilage of the peels of the The solvents acetonitrile  and formic acid  were purchased from Merck . 2,2′-Azobis(2-amidinopropane) (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and cyanidin-3-glucoside were purchased from Sigma-Aldrich .Vaccinium meridionale Sw.) fruits were collected from wild shrubs in the vicinity of the municipality of Raquira, located at 2150 m above sea level  and with an average temperature of 16 °C, in the department of Boyacá, Colombia. The black–violet fruits were selected, washed, chopped, and then homogenized in a blender at minimum power for 1 min. The resulting product was frozen at −80 °C in an ultralow-temperature freezer  for 48 h. The samples were then lyophilized in a 4.5 L FreeZone lyophilizer  at −84 °C under a 0.13 mbar vacuum for 48 h. After lyophilization, the samples were ground in a food processor and stored at a refrigerated temperature (4 °C) in amber bottles until further analysis. Non-microencapsulated lyophilized Andean berry pulp (LABP) was designated as the control sample.Fresh Andean blueberry , and left for 12 h. The hydrated peels were manually squeezed to extract their gel. Then, 95% ethanol at 18 °C was added to the extracted gel at a 3:1 ratio (ethanol:gel), and the mixture was allowed to stand for 15 min without stirring until the formation of a milky-white supernatant corresponding to the fruit peel mucilage. The mucilage was collected and then dried in an oven at 50 °C for 3 h. The dry material was manually macerated in a porcelain mortar and, subsequently, filtered through a 60-mesh sieve until a fine powder was obtained (standard granulometry ≤ 250 µm). The powdered mucilage was placed in high-density polyethylene bags and stored in a desiccator, at room temperature, with a relative humidity of 30%, until characterization.Mucilage was extracted from a et al. . PricklyTwo different microcapsule formulations were prepared according to the design shown in g rpm, 20 min, 4 °C). The absorbance of each solution was measured at 535 nm using a Macherey-Nagel  Nanocolor UV/VIS II spectrophotometer. The total anthocyanin (TA) concentration in the extracts was determined and expressed as cyanidin-3-O-glucoside (C-3-O-G) according to Equation (1). All results were expressed as mg C-3-O-G equivalents/L of samples.A = (absorbance at 535 nm), WM = the molecular weight of C-3-O-G (449.2 g/mol), DF = the dilution factor, V = the total volume of the diluted samples (mL), ε = the molar extinction coefficient of C-3-O-G , and m = the weight of samples (g).The total anthocyanin contents in the microcapsules, obtained on the following day after drying and storage in the dark at room temperature, were determined using the method reported by Garzón et al. , with sov/v) acidified with trifluoroacetic acid (1%) under agitation in a vortex for 45 min, centrifuged at 5000× g rpm for 20 min at 4 °C, and the supernatant was filtered through a 0.45-micron Millipore filter. Aliquots (5 µL) were separated through a PREMIER BEH C18 AX analytical column  . The eluent system was composed of type I water and 0.1% formic acid (solvent A), and acetonitrile and 0.1% acid formic (solvent B), at a flow rate of 0.5 mL/min. The gradient program was 95:5 v/v at 0 min, 80:20 v/v at 10 min, 60:40 v/v at 14 min, and 95:5 v/v at 15 min. The following parameters were maintained: source temperature of 120 °C, desolvation temperature of 500 °C, desolvation gas flow rate of 900 L/h, and cone gas flow rate of 100 L/h. The cone and capillary voltages were set at 40 V and 2.0 kV, respectively.The identification of anthocyanins in the LABP, SD-MP, and SD-GA samples was performed using an Acquity H Class Plus Ultra-High-Performance Liquid Chromatography (UPLC) device coupled a with Xevo-G2-XS Quadrupole Time-of-Flight (QTOF) detector . These UPLC-QTOF analyses were performed in positive electrospray ionization (ESI) mode. For extracting anthocyanin compounds, 35.2; 32.8, and 34.6 mg of LABP, SD-MP, and SD-GA, respectively, were extracted with a methanol:water mixture  was measured using the method reported by Prior et al. . First, The total dietary fiber (TDF) contents in the powdered mucilage and in the SD-MP and SD-GA microcapsules were determined using a total dietary fiber test kit (TDF-100A) provided by Sigma-Aldrich , based on the enzymatic–gravimetric method AOAC 985.29 .L*, a*, and b*) of the powdered mucilage and gum arabic, as well as of the SD-MP and SD-GA microcapsules (samples placed in Petri dishes), were determined randomly on three different surfaces using a Chroma Meter CR-300 , and their intensity was recorded accordingly. The instrument was calibrated with a white tile calibration plate . The results were expressed as the CIE L*, a*, and b* color parameters, where L* represents the lightness, a* represents the redness (+) to greenness (−), and b* represents the yellowness (+) to blueness (−) of the samples.The color attributes (abh) were calculated using Equations (2) and (3), respectively [The derived color parameters of chroma (ectively :(2)Cab*=The particle size distribution, average diameter, and polydispersity index of the SD-MP and SD-GA microcapsules were determined by the laser diffraction technique using a NanoPlus zeta/nanoparticle analyzer . The SD-MP and SD-GA microcapsules were diluted in type I water to adjust the obscuration range as follows: viscosity of 0.8878 cP, average refractive index of 1.33, and sample temperature of 25 °C, in a glass cuvette.The microscopic morphology of the SD-MP and SD-GA microcapsules was evaluated by scanning electron microscopy (SEM) using an EVO MA 10-Carl Zeiss device  operating at 20 kV. All samples were coated using gold–palladium sputtering before their examination.Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of the gum arabic and the SD-MP and SD-GA microcapsules were performed using a TA Instrument . Argon was used as a purge gas (100 mL/min). The dried samples were placed in aluminum pans and heated from 20 to 600 °C at a heating rate of 10 °C/min.n = 3). Data were analyzed using analysis of variance (ANOVA), and means were compared using Fisher’s least significant difference test (p < 0.05).The total anthocyanin contents and antioxidant capacity (ORAC), along with the color parameter data presented in As shown in Opuntia ficus-indica cactus mucilage improved the antioxidant activity of amaranth microcapsules.As expected, the higher retention of anthocyanins in the SD-MP microcapsules gave them a higher antioxidant activity than the SD-GA microcapsules . In factOpuntia ficus-indica cladodes improved the total dietary fiber content of pink guava microcapsules. Therefore, both types of microcapsules could be applied as additives in the development of nutritionally enhanced food products, since their incorporation—for example, in dairy products—could reduce the fat content by adding them in powder form.As also shown in L* = +10.7) than that recorded for the microcapsules formulated with 1.0% OFI mucilage , which was attributed to the whiter color and higher contents of the gum arabic powder compared to the color and contents of the mucilage powder [a* (15 ≤ a* ≤ 25) indicated that the color of both microcapsules was framed in red tones, which is consistent with the presence of anthocyanins in the obtained microcapsules [a* for SD-GA microcapsules  is consistent with the highest retention of anthocyanins [b* was higher for the SD-MP microcapsules (∆b* = +3.6), which coincides with the yellower hue of the OFI mucilage compared to the hue of the gum arabic. Similar results were reported by Ahmada et al. [As shown in e powder . The rancapsules . The higocyanins , as indiocyanins  for pomea et al.  during tabh) of the particles coincided with a pure red hue in the color space, which was lower for SD-GA compared to SD-MP microcapsules. It was observed that the original hue of the gum arabic  and of the mucilage —located in the range from white to yellow—managed to influence the tonality of the encapsulations. Bhagya Raj and Dash [The chroma parameter  and cyanidin-3-O-alpha-arabinopyranoside) constituted the two dominant pigments by far (83.3 ≤ anthocyanin % ≤ 99.6) in the samples. Although the anthocyanin ideain had a slightly higher concentration than cyanidin-3-O-alpha-arabinopyranoside in the unencapsulated sample (LABP), in the SD-MP and SD-GA microcapsules the ideain concentration was reduced compared to its cyanidin analog, indicating a greater thermal susceptibility of the former compared to the latter during the spray-drying process. The anthocyanins peonidin-3-O-alpha-arabinoside, delphinidin-3-arabinoside, and delphinidin-3-pyranoside presented lower but significant concentrations (3.5 ≤ anthocyanin % ≤ 7.4) in the LABP sample; however, their concentrations in both microcapsule samples (SD-MP and SD-GA) were found to be below 1%, possibly indicating greater thermal sensitivity of these natural dyes during the spray-drying process. For its part, the anthocyanin with the highest molecular weight ) maintained its concentration (0.1%) in all samples.The HPLC chromatograms and mass spectra of the two most abundant anthocyanins  found in the Andean blueberry samples LABP, SD-MP, and SD-GA are presented in the Aloe barbadensis Miller) mucilage as a wall material [Opuntia monacantha cactus in the zeaxanthin nanoparticle formulation increased the particle size. Finally, the increase in the particle size of the SD-MP powder was consistent with the presence of anthocyanins and was correlated with thermal stability of the microcapsules  was higher than that of the SD-MD microcapsules (0.54), indicating a fairly narrow size distribution of particles in the latter, suggesting a good solubility  when they are incorporated into a food matrix as a coloring additive, because they will not easily form agglomerates .Opuntia monacantha cactus cladode mucilage as a wall material.SEM micrographs of the SD-MP and SD-GA microcapsules taken at 500× and 5000× magnifications are shown in The thermal behaviors of gum arabic and the SD-GA and SD-MP microcapsules are shown in Opuntia monacantha cactus cladode mucilage as a wall material [Opuntia ficus-indica cactus cladode mucilage as an encapsulating agent [The thermogram of the SD-MP microcapsules b revealematerial , as wellng agent .The thermogram of the SD-GA microcapsules c showed The microcapsules containing mucilage had a significant mass loss around 375 °C in contrast to the microcapsules made up of gum arabic, which had their greatest mass loss at approximately 323.96 °C. This result shows that the SD-MP microcapsule powder has superior thermal stability, possibly due to the higher molecular weight of the hydrocolloid . TherefoOpuntia ficus-indica as an alternative encapsulation material, while the other microencapsulate used gum arabic as a traditional encapsulation material.In this work, a comparative analysis of the properties of two different microcapsules made by spray-drying of Andean blueberry anthocyanins was carried out. One of the microencapsulates used the mucilage extracted from the peels (byproducts) of the prickly pear The microcapsules obtained with OFI mucilage (SD-MP) presented an anthocyanin loading capacity approximately five times higher than that of the microcapsules formed using gum arabic (SD-GA). This difference was associated with the formation of larger spherical microcapsules with a smoother surface and with greater thermal resistance when OFI mucilage was used as structural material. This result was attributed to the greater emulsifying and gelling properties of OFI mucilage compared to gum arabic, allowing a superior thermal protection and stabilization effect on the anthocyanins contained in the SD-MP microcapsules. Consequently, the mucilage microcapsules presented a higher antioxidant capacity than the unencapsulated Andean blueberry pulp (LABP) and that encapsulated with gum arabic (SD-GA), caused by a contributory effect between the high load of anthocyanins and the presence of polyphenols from the OFI mucilage. As expected, the SD-MP microcapsules were characterized by a greater red hue due to their higher anthocyanin contents. Additionally, the structure and relative percentage of anthocyanins present in both the unencapsulated lyophilized sample (LABP) and the SD-MP and SD-GA microcapsules were elucidated using the HPLC-MS/MS technique. Here, cyanidin-derived anthocyanins  mostly dominated the anthocyanin contents in all samples. On the other hand, microcapsules of gum arabic (SD-GA) presented higher contents of total dietary fiber.In summary, it can be seen that the mucilage extracted from OFI prickly pear peels has advantageous properties as an encapsulating material for highly antioxidant pigments, such as Andean blueberry anthocyanins. This condition gives the mucilage microcapsules containing Andean blueberry anthocyanins the potential to be used in the food industry as water-soluble natural colorants for the formulation of novel and functional products (antioxidant + dietary fiber) that can be classified with the “clean label”; however, it is necessary to take into account different aspects that should be deeply investigated in the future, including evaluation of the behavior of the microencapsulates as coloring additives in the processing of model food systems, and their sensory acceptance by consumers, as well as the effects of the microencapsulation on the bioaccessibility and bioavailability of bioactive compounds.The results of this work are a structural part of the National Invention Patent Application No. NC2022/0007738, submitted for evaluation to the Superintendencia de Industria y Comercio of Colombia.Opuntia ficus-indica cactus fruits of Colombian origin.In compliance with the provisions of the Contracts for Access to Genetic Resources and its derivative products No. 364 and 365 of 2022, signed between the Ministerio de Ambiente y Desarrollo Sostenible of Colombia and the Universidad de Boyacá and the Universidad Pedagógica y Tecnológica de Colombia, respectively, it should be clarified that the genetic material  was extracted from


Our findings suggest that the YBT-UQ has limited value as a field-based screening tool to assess the risk of sport-related injuries in adolescent handball players.Handball players are at a high risk of suffering a sport-related injury. Recent studies in various adult populations  showed that poor scores in the upper quarter Y-balance test (YBT-UQ) are related to an increased risk of injury. Yet, it is unclear whether this also applies to adolescent handball players. Thus, the present study aims to determine if pre-season YBT-UQ performance is associated with sport-related injuries during the competitive season in adolescent handball players. One hundred and thirty-three adolescent handball players  who competed in the second highest league in the Rhine-Ruhr region, Germany, during the 2021/2022 season participated in the study. Before the competitive season, the players performed the YBT-UQ to assess upper extremity mobility and stability of the throwing and non-throwing arm. Over the 8-month competitive season, the coaches monitored the occurrence of sports-related injuries once a week, using an injury report form from the legal accident insurance. Fifty-seven players (43%) incurred a sport-related injury during the competitive season, of which 27 (47%) had upper body injuries, and 30 (53%) were lower body injuries. The YBT-UQ performance of the throwing and non-throwing arm did not significantly differ between injured and non-injured players. Further, Cox proportional hazard survival regression model analyses revealed that only the presence of an inferolateral reach asymmetry score ≥7.75% arm length was associated with a moderate increase in the risk (hazard ratio = 2.18, 95% confidence interval = 1.02–4.68,  It is pInjuries have adverse effects, as injured players cannot develop individually and cannot contribute to their team's success . In addiN = 494, m = 454, f = 40; age: 28.6 ± 6.8 years) were reported by Campbell et al. . The odds ratio (OR) to suffer an injury was 5.4 in the case of a CS of 81.8–89.3% AL, and an OR = 3.6 in the case of a CS of 89.0–99.0% AL for the left arm. For the right arm, an OR = 5.4 was present in the case of a CS 77–88.9% AL, and an OR = 3.6 in the case of a CS of 89.0–102.7% AL. The authors concluded that lower scores in the YBT-UQ were associated with an increased injury risk. In the most similar group to handball players, Bennett et al.  non-injured sub-elite adolescent handball players . Before 2.2.2.2.1.2). Training experience in years as well as the dominant arm and the throwing arm, were requested from all participants and recorded by the examiner.Body height, mass, and upper-limb length were assessed before the testing. The upper-limb length was measured from the distal tip of the middle finger, with the shoulder at 90-degree abduction , to the 2.2.2.The YBT-UQ was executed with a Y Balance Test Kit . A specific YBT-UQ testing protocol was used to assess the maximal reach in the different reach directions of each participant. The examiners were either graduates of sports sciences or physiotherapists and were accustomed to executing the YBT-UQ. The correct execution of the tests was demonstrated by one of the examiners before the actual tests were carried out. All subjects had to start in a push-up position  with the2.2.3.All players and their respective coaches agreed to continuous monitoring of injuries. As the YBT-UQ assesses mobility and stability of the whole kinetic chain , and defTo ensure high compliance, coaches who missed submitting the information were actively contacted by the assessor to ensure consistent monitoring of the injury data. To gather all information in a timely manner, an alternative contact person from the coaching staff of each team was identified and contacted in case of delays.2.3.a priori power analysis with G*Power  and the following a priori predictor variables: IL reach asymmetry (two levels: ≥7.75% AL and <7.75% AL); normalized SL reach distance (two levels: ≤80.1% AL and >80.1% AL); absolute SL reach distance (two levels: ≤57.75 and >57.75 cm); normalized CS (two levels: ≤81.1% AL and >81.1% AL). In accordance with Hopkins , which was used for all analyses. The sample size was estimated based on an  G*Power . The ana Hopkins , effect 3.Of the 133 players assessed, all players were tracked over the course of the entire season. There were no dropouts, and only one player changed clubs during the season. However, data on this player were still accessible, as he changed from one club participating in the study to another. Overall, the coaches’ response rate throughout the season was 100%, indicating that all data and weekly reports were provided to the examiner. No significant differences in preseason YBT-UQ performance were found between injured and non-injured players for the throwing and the non-throwing arm reach .p = 0.898; IL: p = 0.854; SL: p = 0.312; CS: p = 0.751). In total, 14 out of the 57 injuries occurred during matches, while the remaining 43 occurred during training sessions. Based on the total match hours of all assessed players , this corresponds to a match injury rate of 4.5 injuries/1,000 match hours.Fifty-seven players (43%) incurred a sport-related injury during the competitive season, of which 27 (47%) were upper body injuries and 30 (53%) lower body injuries . In termp = 0.045] of lower body injuries   .4.The study aims to investigate if preseason YBT-UQ performance is associated with sport-related injuries during the competitive season in adolescent handball players. The main results can be summarized as follows: (a) no significant differences in preseason YBT-UQ scores were found between injured and non-injured players both for the throwing as well as the non-throwing arm; (b) YBT-UQ distance and asymmetry cut-off values (except IL reach asymmetry) could not be confirmed as factors for an increased risk of sport-related injuries in the present target group.Our results align with those of Bennett et al. , who alsSeveral reasons may explain the lack of relationships between YBT-UQ performance and sport-related injuries. The study by Campbell et al.  used a dThe match injury rate of 4.5 injuries/1,000 match hours found in the present study is less than those reported by Olsen et al. , who fou4.1.Different limitations of the study must be addressed. The baseline values of the YBT-UQ were assessed on a single occasion, sometimes referred to as a “static snapshot” . However5.We investigated whether preseason YBT-UQ performance was associated with sport-related injuries during the following competitive season in adolescent handball players. Our results indicate that neither the YBT-UQ reach distances of the throwing or non-throwing arm nor the asymmetry cut-off values (except IL reach asymmetry) were associated with the occurrence of a time-loss injury. Thus, the YBT-UQ seems to have limited benefit as a field-based screening tool for assessment of injury risk in adolescent handball players. Future studies may investigate if the YBT-UQ could be a useful tool when added to a multifactorial test battery. In addition, it would be desirable to include injury mechanisms in terms of contact vs. non-contact, acute vs. chronic, and timing during the games. Moreover, future studies should assess whether the limited association between YBT-UQ results and handball-specific injuries that was demonstrated in our study also applies to other groups of players, i.e., even younger or adult players.


The primary objective was to evaluate the safety of the anti-PD-1 antibody camrelizumab in people living with HIV (PLWH); the secondary objective was to evaluate tumor response.+ T-cell count greater than or equal to 100 cells/µL were treated with camrelizumab in daily practice. We describe the demographic characteristics, safety, and clinical course of these 24 PLWH with cancer treated with camrelizumab. Safety was assessed using the current Common Terminology Criteria for Adverse Events (CTCAE). The tumor response was assessed according to the Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST 1.1).From May 8, 2018, to December 10, 2021, twenty-four patients with HIV and advanced cancer as well as a CD4The median number of cycles was 8 (4–26). Only two grade 3 adverse reactions were reported (no toxic deaths or immune-related deaths). Among the 24 patients, 2 (8%) complete responses and 6 (25%) partial responses were observed. 7 patients (29%) were at stable tumor status and others progressed.Data from the present study strongly support the use of camrelizumab  in this population, as it appears to be a feasible approach with no deleterious effects on PLWH and tolerability and acceptable efficacy. In addition, these findings further support the inclusion of PLWH with cancer in clinical trials evaluating the safety and efficacy of ICIs on cancer. Camrelizumab had an acceptable safety profile in 24 participants with HIV, a CD4+ T-cell count of greater than 100 cells/μL, and advanced cancer. This study increases confidence in immunotherapy for HIV. With the advent of highly active antiretroviral therapy in recent years, people living with HIV (PLWH) have become less likely to die of AIDS-related illnesses. However, non-AIDS-related malignancies have been identified as the leading cause of death in PLWH. Compared with the general population, PLWH are at a higher risk of cancer –3. The tImmune checkpoint inhibitors (ICIs) have emerged as a powerful new tool in cancer treatment. Immunotherapy has received considerable critical attention; however, current research and guidelines lack information on immunotherapy in PLWH advanced cancers . ImmunotThe adverse event (AE) profile of checkpoint inhibitors targeting PD-1、PD-L1 has been tested in the general population with cancer and the immune-related AEs related to anti-PD-1 therapy occur in fewer than 30% of patients with cancer. The most common AEs are with skin, musculoskeletal, gastrointestinal, and endocrine, being generally mild to moderate . Safety This retrospective observational study included patients diagnosed with locally advanced or metastatic cancers who received treatment in our hospital from 8th May 2018 and 10th July 2021. Included cases were followed up by telephone until June 2022. Patients ’clinical data were taken from electronic health records in the hospital information system. The clinical and laboratory data were retrospectively retrieved by telephone and hospital medical case records. This study was subject to approval by the Ethics Review Committee of our hospital.Inclusion criteria were the following: (1) with controlled HIV viral load; (2) the baseline CD4 + T-cell counts > 100 cells/µL; (3) with no central nervous system metastases, (4) with complete data, including general laboratory and radiological data, related oncology treatment data ; (5) with an Eastern Cooperative Oncology Group (ECOG) performance score of ≤ 1;(6) not AIDS-defining cancers.+ T-cells count, HIV viral load before immunotherapy, and the number of cycles received. Camrelizumab (200 mg) wea administered intravenously every 2 weeks until death or intolerability. Treatment continued until disease progression, unacceptable toxicity, patient withdrawal, or investigator decision. The tumor response outcome was assessed by the radiographic data according to Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST 1.1). The adverse events were collected by the follow-up according to the Common Terminology Criteria for Adverse Events, version 4.03.A clinical physician collected data available on the case report form, including characteristics, baseline CD4Changes in CD4 + T-cell count from baseline to the time of last treatment cycle were evaluated by Wilcoxon signed rank test. The cut-off time for follow-up was until May 2022, after the completing the last cycle or death. SPSS (version 26) were used for statistical analysis.The primary objective was to assess the safety and tolerability of camrelizumab in patients with HIV on ART with locally advanced or metastatic cancer. Immune-related AEs of grade 2 or higher were considered immune-related events of clinical interest. The secondary objective was to produce preliminary insights into clinical benefits. Tumor response was assessed by clinical trial group criteria, and clinical benefit was defined as complete response (CR) and partial response (PR).+ T-cell count (< 100 cells/µL), and 8 were in other clinical trials, were excluded. A total of 24 patients were included in this study. . Twenty-three (96%) received ART before the Anti-PD-1 therapy. No change in HIV viral load was changed during follow-up. CD4+ T-cell counts were monitored during treatment in 13 patients. Figure + T-cell counts in 13 patients who had value under camrelizumab. There is no significant change in CD4+ T-cell count between the baseline and the time of the last treatment (p = 0.786). Overall, there was no obvious change.From May 8, 2018, to December 10, 2021, 314 PLWH with cancer have been presented. Thirty-six patients were proposed for Anti-PD-1 therapy, 2 selected other anti-PD-1 medicine, 2 with a low CD4Safety was noted over the course of 207 cycles in 24 participants. The median number of cycles was 8 . At the time of analyses, Five participants continued to receive Anti-PD-1 therapy. Treatment-emergent AE s at least possibly attributed to Camrelizumab that occurred in participants are presented in Table The tumor types and response were presented in Table In the last few years, cancer immunotherapy has significantly advanced. Immune checkpoint proteins, like PD-1, have taken an active role in mediating T-cell exhaustion both in cancer and chronic infections. Upon interaction with their ligands, for example, PD-L1, a negative signaling cascade is activated within the T cell that blocks T cell receptor-mediated activation. Consequently, the T cell loses its effector function, and thus, its ability to destroy cancer cells or provide cytokines for proper immune homeostasis. Once the interaction between the checkpoint protein and its ligand is interrupted, for example, by a blocking antibody, T-cells can regain effector functions and participate again in cancer surveillance . Anti-PD+ T-cell.In this study, CD4 T-cell count levels were found to fluctuate, slightly increasing or decreasing, and at the last therapy cycle, there were 3 patients with levels that were lower than the baseline level. Our results showed that camrelizumab had no detrimental effect on CD4 T-cell counts. The previous study . found CThe most common AE was reactive capillary endothelial proliferation (RCEP) (238 [79.9%]. vs. 32 [10.8%].) in the previous study, this adverse event mainly occurred at grade 1 or 2 . Most paData on the tolerance and efficacy of Anti-PD-1 in PLWH are scarce. To the best of our knowledge, this is the first study that evaluated camrelizumab in PLWH with advanced cancers. Previous studies assessed the safety of pembrolizumab in PLWH and advanced cancers, and tumor regression in participants with a range of tumor types confirmed the activity of anti-PD-1 therapy in PLWH . Spano eThe retrospective study design and sample size did not allow for a formal comparison of rates of specific AEs in the general population. In addition, this study was not randomized. Although this study demonstrated camrelizumab had a clinical benefit in PLWH with several cancers, this study did not have enough participants with a tumor to evaluate the tumor response accurately.Data from the present study strongly support the use of camrelizumab  in this population, as it appears to be a feasible approach with no deleterious effects on PLWH and tolerability and acceptable efficacy. In addition, these findings further support the inclusion of PLWH with cancer in clinical trials evaluating the safety and efficacy of ICIs on cancer.


Inverse restricted kinematic alignment (irKA) was modified from restricted kinematic alignment for total knee arthroplasty (TKA). This prospective single-center study aimed to evaluate the outcomes of irKA-TKA on all knee subtypes classified by Asia specific (Huang’s) phenotypes. A total of 96 knees that underwent irKA-TKA at one hospital between January 2018 and June 2020 were included, with 15 knees classified in Type 1, nine in Type 2, 15 in Type 3, 47 in Type 4, and 10 in Type 5 by Huang’s phenotypes. Outcomes were knee alignment measures and patient-reported satisfaction evaluated by the Western Ontario and McMaster Universities Arthritis Index (WOMAC) and traditional Chinese version of the Forgotten Joint Score-12 (FJS-12). Follow-up was one year. Type 4 knee was most significantly corrected in all angles by irKA-TKA, followed by Type 2 and 3 knees. Type 5 and 1 knee were only significantly corrected in some angles. The correlation between FJS-12 and WOMAC was good at 6 months (Pearson correlation coefficient (r) = 0.74) and moderate at 6 weeks, 3 months, and 12 months during follow-up (r = 0.37~0.47). FJS-12 and WOMAC displayed comparable hip–knee–ankle angle cut-off value (4.71° vs. 6.20°), sensitivity (70.49% vs. 67.19%), specificity (84.00% vs. 85.71%), and Youden index (54.49% vs. 52.90%) in prediction of good prognosis. In conclusion, irKA-TKA corrects knee alignment in all knee types with increasing satisfaction for one-year follow-up. Knees with presurgical varus deformity are most recommended for irKA-TKA. Both presurgical scores of the traditional Chinese version of FJS-12 and WOMAC predict the prognosis of irKA-TKA. Osteoarthritis is a debilitating condition that causes disability, psychological stress, and poor quality of life. Total knee arthroplasty (TKA) is a frequently used orthopedic procedure to treat severe osteoarthritis and improve a patient’s mobility and well-being. Singh et al.  used datInverse restricted kinematic alignment (irKA), a tibia-first and gap balancing version of rKA, was developed for excellency ,11. A prPatients older than 60 years old and who received inversed restricted kinematic alignment (irKA) total knee arthroplasty (TKA) between January 2018 and June 2020 in the China Medical University Hospital were included for this study. Patients with revision surgery, previous trauma around the knee, knee collateral ligament injury, pre-knee malignancy, and losing follow-up within 1 year were excluded.All the patients had long-leg standing X-rays radiographs according to the protocol suggested by Ramadier et al.  and SaraThe frontal plane was first accurately determined by looking for a true lateral view of the knee. This was done by rotating the posture until the posterior margins of the condyles were superimposed. While maintaining the X-ray source perpendicular to the frontal plane, the image intensifier was turned 90° around the knee to obtain an accurate long-leg AP standing view. The footprint was drawn on cardboard to reproduce the same orientation of the lower leg pre- and postoperatively. The same view can be reached by placing the foot on the cardboard afterward. The HKAA, KAA, LDFA, and MPTA could then be measured from the long-leg film.This protocol was used for all the patients to avoid any errors in the measurements related to the rotation of the lower leg. The measurements were done with a ruler and a manual goniometer by one radiologist and one orthopedic practitioner independently.On the day of the surgery, a liquid or semi-liquid diet was provided. To reduce bleeding during the surgery, one gram of tranexamic acid  dissolved in 100 mL of 0.9% NaCl was infused intravenously thirty minutes before the operation.® NexGen® LPS Fixed Knee system . The tibia resection was conducted first to restore the pre-arthritic MPTA within the safe zone. The restricted safe zone was defined as 86° to 93° for recreation of both the LDFA and the MPTA, and −5° varus to +4° valgus for the final HKAA and KAA. A medial parapatellar arthrotomy approach was chosen. The tibial cut was aimed to reconstruct the native tibial joint line obliquity. Then, after tibial cut  for 4 weeks after surgery. The first follow-up appointment was 6 weeks after surgery.The knee orientation was measured in terms of HKAA, KAA, LDFA, and MPTA to determine whether the native limb and knee alignment was restored to the natural physiological state after irKA-TKA. The HKAA, KAA, LDFA, and MPTA were measured from the films of the X-ray radiography taken at 6 weeks follow-up.Patients were asked to evaluate the pain or discomfort in their daily life before and after irKA-TKA surgery by the Western Ontario and McMaster Universities Arthritis Index (WOMAC) and the traditional Chinese version of Forgotten Joint Score-12 (FJS-12). WOMAC and FJS-12 were conducted by questionnaire to quantify the severity of knee pain with daily activities at 6 weeks, 3 months, 6 months, and 12 months after surgery. The WOMAC was also reported before surgery for comparison.This was a longitudinal prospective cohort study of a single surgeon series registered at clinicaltrials.gov (NCT05685706) and performed in accordance with the declaration of Helsinki. The study protocol was reviewed and approved by the China Medical University and Hospital Research Ethics Committee (CMUH110-REC2-030). Written informed consent was signed by all the participating subjects.n (%) using Fisher’s exact test. The distributions of LDFA/MPTA before and after operation were presented in frequency histograms. The changes of HKAA, KAA, LDFA, and MPTA before and after operation were examined by Wilcoxon sign-rank tests. Trend tests for the improvement in the FJS-12 and WOMAC scores were calculated from simple linear regression models with the FJS12/WOMAC scores as a response variable and the order of time course as a predictor. The consistency of FJS-12 and WIMAC for each time point was performed by Spearman correlation analysis. The sensitivity and specificity of predicted results were calculated to make a receiver operating characteristic (ROC) curve. The cutoff point of HKAA before operation was decided by the maximum value of the Youden index. Each area under the curve (AUC) was calculated, with a higher AUC indicating higher predictive performance. We extracted the maximum AUC to define the prognosis by the FJS-12 and WOMAC score and HKAA before operation as the independent variable using univariate logistic regression. If a patient had a prognosis FJS-12 score ≥ 72.9 or WOMAC score ≥ 82, then the patient will be considered to have good prognosis. Using the FJS-12 ≥ 72.9 or WOMAC score ≥ 82 as a good prognosis, we identified the cutoff point of HKAA before operation to predict the prognosis of our patient. All statistical analyses were performed by using SAS software . A two-sided p < 0.05 was established as statistically significant.As for the comparison between five types of phenotypes, continuous data with normal distribution were presented as mean ± standard deviation (SD) using Student’s test and data without normal distribution were presented as the median and interquartile range (IQR: 25th—75th percentile) using the Wilcoxon rank sum test. Categorical data were presented as In total, 76 patients with 96 knees were included in this study. It included 60 females (75 knees) and 16 males (21 knees). The mean age was 70.4 ± 9.7 years old. For the knee classification, 15, 9, 15, 47, and 10 knees were grouped into Type 1, 2, 3, 4, and 5, respectively. The baseline information of the study population is presented in Distributions of LDFA/MPTA before and after surgery are presented as the frequency histograms in p < 0.001, p = 0.006 and p = 0.009) and MPTA increased (p < 0.001) in the whole study population. In Type 1, HKAA was the only parameter that changed significantly . In Type 2 and 3, HKAA in each group reduced significantly (p = 0.027 and 0.003) and LDFA and MPTA significantly increased. In Type 4, HKAA, KAA, and LDFA significantly reduced  and MPTA increased . In Type 5, LDFA significantly increased  and MPTA reduced .p < 0.001) and the significance of valgus was not found (p = 0.068) because of the reduced mean score from the 3th month to 6th month (83.33 to 63.54). WOMAC had a significant increasing trend from pre-op to the 12th month in each type . Neutral and varus knees had the largest increase from pre-op to the 6th week, specifically. Comparing FJS-12 and WOMAC at each follow-up time, the significant positive correlations were found at the 6th month  and 12th month .The receiver operating characteristic (ROC) curve of HKAA is shown in Our study indicated that irKA-TKA restored the pre-arthritic MPTA, LDFA, and HKAA in all knees classified by Huang’s phenotypes. The FJS-12 and WOMAC showed a similar trend of change with increasing satisfaction within the 12 month follow-up. The correlations between FJS-12 and WOMAC was good at 6 months (r = 0.74) and moderate at 6 weeks, 3 months, and 12 months  as a measure of the Pearson correlation coefficient.irKA is a tibia-first and gap balancing version of rKA, which is derived from KA and MA. rKA increased the ratio of satisfaction from 81% patients in MA to 93% . AdditioAnother study determined the threshold of the FJS-12 for detecting the patient acceptable symptom state (PASS) following primary TKA . They reIn this study, we predicted that HKAA higher than 6.20° or 4.71° had a good prognosis by WOMAC and FJS-12, respectively. Another study reported that patients with preoperative HKAA more than 10 degrees varus had a better outcome when receiving KA than MA . Thus, iThe purpose of TKA is restoring the native harmony of the knee. Three interdependent elements, including morphology, soft-tissue balance, and alignment, should be considered at the same time to reach this goal ,30. As sThere are some limitations in this study. First, the sample cohort was small. This made it difficult to generalize the finding. Second, the timeframe of follow-up was only 2 years. The conclusion may be challenged in long-term conditions. Third, there was no control group in our study. A comparison between mechanical, kinematic, or anatomic alignment in TKA would create an observation with more significance. Fourth, this was a prospective study based on a single surgeon. The protocol, custom, and facility may affect the results unintentionally.irKA-TKA correct knee alignment in all knee types with increasing satisfaction during one-year follow-up. The traditional Chinese version of FJS-12 was moderate or well correlated with WOMAC during follow-up. The presurgical scores of both FJS-12 and WOMAC can predict prognosis after irKA-TKA. Knees with preoperative varus deformity are most recommended for irKA-TKA.


Ex-PRESS glaucoma shunt stainless steel devices have been used worldwide for glaucoma treatment. The purpose of this study was to evaluate the safety of high-field magnetic resonance imaging (MRI) for Ex-PRESS–inserted eyes.Using rabbits, we performed Ex-PRESS shunt surgery in one eye in each rabbit and divided the rabbits into MRI and non-MRI groups. In the MRI group, 1 week after Ex-PRESS shunt surgery under low specific absorption rate (SAR) conditions and 1 week later under high SAR conditions, high-field 4.7-Tesla MRI was performed. Aqueous flare counts were measured before and after the Ex-PRESS shunt surgery and each MRI examination. The rabbits in the non-MRI group received only general anesthesia, and aqueous flare counts were measured as for those of the MRI group. Aqueous flare counts were expressed in photon counts per millisecond.P = 0.01). The day after MRI, the flare count improved spontaneously to the same level as that before MRI.No dislocation of the Ex-PRESS shunt device was observed after MRI. The flare count ratio (MRI/non-MRI) in Ex-PRESS–inserted eyes 2 hours after high SAR MRI increased significantly compared with that before MRI (0.8 ± 0.3 vs 2.7 ± 0.8; pre-high SAR MRI vs 2 hours after high SAR MRI, respectively; Our results indicate that high-field MRI can be performed relatively safely after Ex-PRESS shunt surgery.This study demonstrates the safety of high-field MRI for Ex-PRESS–inserted eyes using a rabbit model. The same observations and examinations were performed for both eyes in the rabbits in each group. The animals were housed and treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures were performed in accordance with the guidelines of the Committee on Animal Experimentation and the Committee of Research Facilities for Laboratory Animal Science, as well as the Natural Science Center for Basic Research and Development of Hiroshima University. All animals in this study were housed for at least 7 days before the experiment in an animal housing facility with a room temperature of 23°C and a 12-hour light/dark cycle (lights on at 8:00 am).Six pigmented male rabbits  were used for the experiments. Ex-PRESS shunt surgery was performed on only one eye of each rabbit. The rabbits were divided into two groups . The rabbits were sedated with an intramuscular injection of 40 mg/kg medetomidine and 1 mg/kg butorphanol. Anesthesia was maintained after intravenous catheterization with an intravenous injection of 50 mg/kg pentobarbital. The right eye was cleansed with topical 0.5% povidone-iodine, and a lid speculum was placed to hold the lids open. A 7-mm conjunctival incision was made along the superior corneal limbus with spring microscissors to make a fornix-based conjunctival flap. A 25G needle was inserted 1.2 mm from the corneal limbus into the anterior chamber, underneath the conjunctival flap. The Ex-PRESS shunt device was then placed in the anterior chamber through the ostium created by the needle. After fixation of the Ex-PRESS device, the conjunctival peritomy was closed using 10-0 nylon with wing sutures. Ofloxacin eye ointment  was applied at the end of the surgery, and no additional medications were administered postoperatively. The procedure required approximately 1 hour, including induction of anesthesia.All experiments were performed with a 4.7-T superconducting magnet system  with a transmit/receive orthogonal volume coil (154 mm inner diameter). The rabbits underwent rapid acquisition of refocused echo imaging  under the two conditions shown in Radiofrequency exposure produces an electric current in the body, creating a thermal effect owing to the absorption of electromagnetic wave energy. SAR is a measure of this thermal effect, usually indicated in watts per kilogram.Before the MRI examination, the rabbits were sedated with an intramuscular injection of 40 µg/kg medetomidine and 1 mg/kg butorphanol. Five minutes after sedation, the rabbits were masked and immobilized with isoflurane at 100 mL/min for 3 minutes in the surgical preparation room. The rabbits were then moved to the MRI room, and anesthesia was maintained with inhalational isoflurane at 60 mL/min. Inhalational anesthesia was maintained for 30 minutes for imaging under low SAR conditions and for 80 minutes under high SAR conditions. In the non-MRI group, anesthesia was administered at the same doses and concentrations as those in the MRI group and MRI was not performed.Aqueous flare was quantified by photometric measurements using a laser flare meter . Aqueous flare counts were expressed in photon counts per millisecond. The rabbit eye was placed in the center of the measurement section with no mydriasis and manually suppressed. Measurements were taken in an area measuring 0.3 × 0.5 mm at approximately the center of the anterior chamber. Measurements were taken by the same examiner; five measurements were taken, and the average value was used in the analysis.am, and the Ex-PRESS implant surgery was performed at noon. Aqueous flare was measured 2 hours after surgery and at 1:00 pm on postoperative days 1 and 2. One week after the surgery, after flare measurement at 1:00 pm, MRI was performed under low SAR conditions. Aqueous flare was measured 2 hours after a low-SAR MRI and at 1:00 pm on days 1 and 2 after a low-SAR MRI. One week after low-SAR MRI, after flare measurement at 1:00 pm, MRI was performed under high-SAR conditions. Aqueous flare was measured 2 hours after high-SAR MRI and at 1:00 pm on days 1 and 2 after high-SAR MRI. In the non-MRI group, flare measurements were taken at the same times as those for the MRI group, but without performing MRI.Preoperatively, aqueous flare was measured at 11:00 t-test. All statistical analyses were performed using JMP pro 16.0.0 , and a P value of less than 0.05 was considered statistically significant.The data were expressed as mean ± standard deviation. Non–Ex-PRESS-inserted eyes acted as controls. To analyze the effect of MRI scanning on aqueous flare counts, we compared the MRI group/non-MRI group ratios for the aqueous flare counts in each of the Ex-PRESS and non–Ex-PRESS-inserted eyes. The time course changes in aqueous flare were compared by Dunnett's test using pre-MRI values as a reference. The aqueous flare count ratios between the Ex-PRESS and non–Ex-PRESS-inserted eyes at each time point were compared by the Student The Ex-PRESS glaucoma filtration device was inserted into one eye of each of the three rabbits in the MRI and non-MRI groups. A photograph of the anterior segment of the eye after Ex-PRESS insertion is shown in P < 0.01). The flare counts in the Ex-PRESS eyes decreased spontaneously to 85.9 ± 78.9 pc/ms and 22.4 ± 12.2 pc/ms on days 1 and 2 after surgery, respectively. In the non–Ex-PRESS-inserted eyes, the flare counts ranged from 10.3 ± 3.2 pc/ms to 12.2 ± 2.4 pc/ms, with no significant change between the preoperative and postoperative day 2 values.Changes in flare counts before and after Ex-PRESS shunt surgery are shown in P = 0.01). Flare count ratios decreased the day after low-SAR MRI and were not significantly different compared with pre–low-MRI ratios. The mean flare count ratio 2 hours after the low-SAR MRI in the non–Ex-PRESS-inserted eyes also increased compared with that of pre–low-SAR MRI . There was no significant difference in the flare count ratios between the Ex-PRESS and non–Ex-PRESS-inserted eyes at all time points (Changes in flare counts and ratios before and after MRI are shown in e points A.P = 0.01). The flare count ratios decreased spontaneously the day after the high-SAR MRI. In contrast, there was no significant increase in flare count ratios after a high-SAR MRI in the non–Ex-PRESS-inserted eyes. Although the mean flare count ratio in the Ex-PRESS eyes 2 hours after a high-SAR MRI was approximately twice as high as that in the non–Ex-PRESS-inserted eyes, this difference was not statistically significant  (P = 0.50).The mean flare count ratio in the Ex-PRESS eyes 2 hours after MRI under high-SAR conditions increased significantly compared with that before high-SAR MRI  B. The meIn this study, we showed that, in Ex-PRESS–inserted eyes, the aqueous flare counts in the anterior chamber 2 hours after MRI increased with high-field MRI. Flare counts decreased spontaneously the day after MRI, and there were no findings indicating Ex-PRESS dislocation after high-field MRI.,,It has been shown that there was no deviation of the Ex-PRESS inserted in human cadaver eyes in a 3-T magnetic field.In this study, the anterior chamber flare counts were increased 2 hours after low-SAR MRI in the non–Ex-PRESS-inserted eyes, although less than those in the Ex-PRESS–inserted eyes. If MRI itself might be unsafe for the eye, the flare counts should still have been increased 1 and 2 days after MRI. However, there was no increase 1 and 2 days after MRI, only after 2 hours. Although the cause of this observation is unclear, the interaction between general anesthesia with isoflurane and MRI may be a potential cause. A study of pediatric patients who underwent MRI showed that serum IL-1β, a marker of systemic inflammation, was significantly elevated after 60 minutes of anesthesia with isoflurane.In this study, we investigated the safety of 4.7-T high-field MRI of Ex-PRESS–inserted eyes and noninserted eyes under two conditions: the low-SAR condition, which is SAR in accordance with the recommendations of international standards and safety guidelines, and the high-SAR condition, which is much higher than the guidelines recommendations. The results showed that intraocular inflammation was induced 2 hours after high-field MRI; however, this inflammation resolved spontaneously the next day without therapeutic intervention, such as with anti-inflammatory drugs. This temporary inflammation may have been influenced by inhalational anesthesia with isoflurane. However, inhalational anesthesia with isoflurane is not usually used in human MRI. This imaging is performed under awake conditions in adults who can remain at rest, and under oral or intravenous sedation in children and adults who cannot remain at rest. No deviation of the Ex-PRESS implants occurred during MRI imaging in either the low- or high-SAR conditions. This suggests that high-field MRI can be performed relatively safely in eyes implanted with Ex-PRESS shunt devices. MRI provides good visualization of soft tissues and is very useful for the early detection, diagnosis, and treatment of systemic diseases. Our study demonstrated the safety of high-field MRI in animal model after Ex-PRESS shunt surgery. This study suggests that high-field MRI imaging at 4.7 T could be relatively safe after Ex-PRESS shunt surgery.


Children tend to have milder forms of COVID-19 than adults, however post-acute complications have been observed also in the paediatric population. In this study, we compared COVID-19-related outcomes and long-term complications between paediatric and adult patients infected by SARS-CoV-2.The study is based on individuals enrolled from October 2020 to June 2021 in the DECO COVID-19 multicentre prospective study supported by the Italian Ministry of Health (COVID-2020–12371781). We included individuals with RT-PCR -confirmed SARS-CoV-2 infection, who were evaluated in the emergency department and/or admitted to COVID-dedicated wards. The severity of SARS-CoV-2 infection was compared across age groups  through the relative risk (RR) of severe COVID-19. Severity was defined by: 1) hospitalization due to COVID-19 and/or 2) need or supplemental oxygen therapy. RR and corresponding 95% confidence intervals were estimated using log-binomial models.The study included 154 individuals, 84 (54.5%) children/adolescents, 50 (32.5%) young/middle-aged adults and 20 (13%) older adults. Compared to young/middle-aged adults the risk of hospitalization was lower among paediatric patients  and higher among older adults . The RR of supplemental oxygen was 0.12 (95% CI: 0.05–0.30) among children/adolescents and 1.46 (95% CI: 0.97–2.19) among older adults. Three children developed multisystem inflammatory syndrome (MIS-C), none was admitted to intensive care unit or reported post-acute Covid-19 complications.Our study confirms that COVID-19 is less severe in children. MIS-C is a rare yet severe complication of SARS-CoV-2 infection in children and its risk factors are presently unknown. COVID-19, the disease resulting from SARS-CoV-2 infection, has affected over 606 million people of whom approximately 6.5 million have died [The spectrum of the disease in adults is well described, ranging from asymptomatic infection to respiratory failure and death. Disease severity increases with age, with consistently higher hospitalization and mortality rates in the older population –4. ComorOn the other hand, in the paediatric population, SARS-CoV-2 infection tends to be asymptomatic or mild, often limited to the upper respiratory tract, and persistent symptoms or long-term sequelae are rarely observed –9. InforThis study aims to compare COVID-19 outcomes, including possible long-term sequalae, across different age groups, in order to further characterize the burden of the disease in children.The study is based on patients enrolled in the DECO COVID-19 project, a multicentre prospective study supported by the Italian Ministry of Health (COVID-2020-12371781), which involved 2 Italian tertiary care paediatric hospitals: 1) Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico in Milan (coordinating centre) and 2) Bambino Gesù Children's Hospital in Rome. The study was approved by the Ethics Committees of both hospitals and informed consent was obtained from the patient or parents/caregivers before inclusion in the study.Mycoplasma pneumoniae and Chlamydia pneumoniae in patients with respiratory symptoms  on a nasopharyngeal swab were enrolled. Demographic and clinical data were collected, including sex, age, body mass index (BMI), comorbidities, maintenance therapy, presence of acute symptoms and their duration, need of hospitalization, admission to intensive care unit (ICU), need of oxygen supplementation and non-invasive or invasive ventilation. In light of the fact that our study was based on PCR confirmed cases of SARS-CoV-2 infection, considering that viral shedding could continue long after the acute phase of the infection, particularly in the pre-vaccine era in which the study was set, we performed an accurate differential diagnosis in all positive patients presenting with acute symptoms suggestive of COVID-19. This included the assessment of an epidemiological link (close contact with subjects with proven active SARS-CoV-2 infection in the familiar/scholastic/working environment 2 to 14 days prior to symptom onset) and the exclusion of other possible causes of infection, by obtaining a nasopharyngeal swab for RT-PCR identification of the most common respiratory viruses, We also gathered information on the complications related to SARS-CoV-2 infection, with a particular focus on multisystem inflammatory syndrome in children (MIS-C). Diagnosis of MIS-C was made according to the Royal College of Paediatrics and Child Health criteria . MacrophInformation on vital status at 6 months after the RT-PCR positive test was also registered.Input of data was accomplished using the REDCap electronic data capture tool, hosted by Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico.We compared the severity of SARS-CoV-2 infection across the following three age groups: children/adolescents aged < 18 years, young/middle-aged adults aged 18–64 years and older individuals aged ≥ 65 years. Severity of COVID-19 was defined according to the following criteria: 1) need of hospitalization due to COVID-19 and/or 2) requirement or increased need of oxygen therapy.Log-binomial models were used to estimate the relative risk (RR) of severe COVID-19 and corresponding 95% confidence intervals (CI) across age groups, using individuals aged ≥ 65 years as the reference category.We enrolled 154 patients with RT-PCR-confirmed SARS-CoV-2 infection. Median age of the study population was 27 years (range: 1 month-89 years) and the study included 84 children/adolescents, 50 young/middle-aged adults and 20 older adults. The most frequently SARS-CoV-2 identified variants were lineage B.1.1.7 (42.4%) and B.1.177 (40.7%).Table The majority of children (56%) was asymptomatic, compared to 18% of young/middle-aged and 10% of seniors. The most frequent symptoms of SARS-CoV-2 infection were fever and cough in all age groups, while dyspnoea, anosmia and/or dysgeusia were almost specific of adult age.Mycoplasma pneumoniae and Chlamydia pneumoniae identification on a nasopharyngeal swab. At least a second viral agent in addition to SARS-CoV-2 was identified in 7 patients, of which 6 had Rhinovirus A/B/C and one had multiple viral coinfection (Rhinovirus and Adenovirus). No case of SARS-CoV-2 and Mycoplasma pneumoniae coinfection was identified in our series. Five of the 7 patients with documented viral coinfection required hospitalization and 4/7 needed oxygen therapy. All patients with multiple viral infections had a positive outcome, making a full recovery.Twenty-seven out of 154 patients, all belonging to the paediatric age group, were tested for possible coinfections, by performing RT-PCR viral, None of the paediatric patients had persistent symptomatic COVID-19 after 2 months from infection, while symptoms lasted longer than 2 months in 6 adults (3 in the 18–64 years age group and 3 aged ≥ 65 years).Hospitalization was required in 27.4% of the children, 56% of young/middle-aged individuals and in 85% of older patients  and higher among those aged ≥ 65 years RR: 1.52) No intensive care unit admission or death due to COVID-19 was registered in the paediatric age group, while 4 adults (1 aged 58 years and 3 aged ≥ 65 years) required intensive care.P < 0.0003).The length of stay was significantly shorter among paediatric patients , as compared to adults . All the deceased had 3 or more pre-existing diseases including diabetes, systemic hypertension, heart disease, liver disease, nephropathy and lung transplantation, which were often the main cause of death.Our study confirms that COVID-19 is generally less severe in children, as indicated by lower rate of hospitalization and need of oxygen therapy. However, in a minority of children (~ 4% of our series) it may present with a severe phenotype, including MIS-C and MAS.In our population, asymptomatic infections were more frequent in the paediatric age group than in adults: 56% of children, in fact, had no symptoms related to SARS-CoV-2 infection as compared to 18% in the young/middle-aged group and 10% in the older group. The rate of asymptomatic infection so far reported in children is quite variable, ranging from 15.5 to 65% –16: thisIn agreement with previous reports, fever and respiratory symptoms were the most common clinical features both in the paediatric and adult population , 18, thoThe use of a diagnostic algorithm applied to the whole paediatric cohort enabled to assess the role of coinfections. Although documented only in a minority of patients exclusively belonging to the paediatric age, viral coinfections were associated with a greater burden of disease, including the need for hospitalization and oxygen supplementation. Though limited, data from our series suggest that coinfections may be associated with a more severe disease course also in children, and thus should be investigated in all patients with a diagnosis of SARS-CoV-2 infection.Comorbidities, particularly diabetes, obesity and hypertension, are known risk factors for severe COVID-19 in adults , 4. In oThe length of hospital stay was significantly lower in the paediatric group, as compared to the young/middle aged and older groups; this finding is consistent with a retrospective analysis of subjects from the early phase of the pandemic conducted in China, which documented significantly shorter hospital stay for COVID-19 in children than in adults . None ofNone of our paediatric patients suffered from long-COVID, according to the National Institute for Healthcare Excellence definition, which includes both ongoing (4 to 12 weeks) and post-COVID-19 (≥ 12 weeks) symptoms . HoweverMIS-C is a multisystem inflammatory syndrome affecting patients aged 0 to 19 years and related to SARS -CoV-2 infection either at the time of diagnosis or in the preceding 2 to 6 weeks. The most frequent clinical features of MIS-C include fever, mucocutaneous findings, cardiac involvement , gastrointestinal symptoms, lymphadenopathy, and neurological manifestations. The exact incidence of this condition is unknown, and at present it is considered a rare complication of SARS-CoV-2 infection . Due to The main limitation of our study is the relatively small size of the enrolled population. This is mainly due to the fact that the present analysis was conducted in the context of a wider project, sponsored by the Italian Ministry of Health. Patients with cystic fibrosis, bronchiectasis and lung transplantation with acute SARS-CoV-2 infection were enrolled over a short period of time and their severity of disease and outcome was compared with the general population. In addition, since the study population was enrolled before the onset or at the earliest stage of the vaccination campaign in our country, we could not evaluate the impact of SARS-CoV-2 vaccination on disease severity and outcome. Finally, data were collected in the pre-Omicron phase of the pandemic, when circulating variants caused a more severe disease than the one secondary to Omicron.However, the fact that we considered only RT-PCR confirmed cases, the search for coinfection and the evaluation of their impact on the severity of disease, the use of objective parameters to assess disease severity and the availability of a 6-month follow up for all the included patients represent significant strengths of our study.This study confirms that COVID-19 is generally less severe in children than in adults. While fever and respiratory symptoms are the most common clinical manifestations in all age groups, symptoms in the paediatric population tend to be milder and often related to inflammation limited to the upper airways, without long-term sequelae. MIS-C is a rare yet severe complication of SARS-CoV-2 infection in children, whose risk factors need to be further defined.Although the SARS-CoV2 pandemic is currently in a resolving phase, the expertise acquired in the assessment of patients in a pandemic setting and the diagnostic algorithm we have used for the present study could represent a useful tool in the management of possible future infectious outbreaks.


Plasmodium falciparum and non-falciparum malaria infections. A laboratory evaluation was performed to assess the performance of the mini-dbPCR-NALFIA for diagnosis of pan-Plasmodium and P. falciparum infections in whole blood.Point-of-care diagnosis of malaria is currently based on microscopy and rapid diagnostic tests. However, both techniques have their constraints, including poor sensitivity for low parasitaemias. Hence, more accurate diagnostic tests for field use and routine clinical settings are warranted. The miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative, easy-to-use molecular assay for diagnosis of malaria in resource-limited settings. Unlike traditional molecular methods, mini-dbPCR-NALFIA does not require DNA extraction and makes use of a handheld, portable thermal cycler that can run on a solar-charged power pack. Result read-out is done using a rapid lateral flow strip enabling differentiation of Plasmodium-positive blood samples from returned travellers (n = 29), and Plasmodium-negative blood samples from travellers with suspected malaria (n = 23), the Dutch Blood Bank (n = 19) and intensive care patients at the Amsterdam University Medical Centers (n = 16). Alethia Malaria (LAMP) with microscopy for species differentiation were used as reference. Limit of detection for P. falciparum was determined by 23 measurements of a dilution series of a P. falciparum culture. A fixed sample set was tested three times by the same operator to evaluate the repeatability, and once by five different operators to assess the reproducibility.Diagnostic accuracy of the mini-dbPCR-NALFIA was determined by testing a set of P. falciparum was 10 parasites per microlitre of blood. The repeatability of the assay was 93.7%  and reproducibility was 84.6% .Overall sensitivity and specificity of the mini-dbPCR-NALFIA were 96.6%  and 98.3% . Limit of detection for Plasmodium infections in whole blood and differentiation of P. falciparum. Incorporation of a miniature thermal cycler makes the assay well-adapted to resource-limited settings. A phase-3 field trial is currently being conducted to evaluate the potential implementation of this tool in different malaria transmission areas.Mini-dbPCR-NALFIA is a sensitive, specific and robust method for molecular diagnosis of  Plasmodium species, its sensitivity for low parasite densities is limited and adequate slide reading requires extensive training and experience [P. falciparum strains with a genetic deletion for the antigen targeted by RDTs, histidine-rich protein 2 (HRP2). Over the past decade, this genotype has become widespread in South America, and increasing prevalence has now been reported for African and Asian countries as well [Correct and timely diagnosis of malaria is key in the management and control of this disease. Traditionally, microscopy of Giemsa-stained thick and thin blood film has been the standard diagnostic technique applied in endemic settings. Although it is able to differentiate the causative perience –4. The dperience . Howeverperience –8. False as well –12. Conv as well , 14.Plasmodium species differentiation [The limitations of microscopy and RDTs can be overcome by the use of nucleic acid amplification techniques (NAATs) . Examplentiation .Plasmodium species. An innovative assay has recently been developed to meet these requirements: the miniature direct-on-blood PCR nucleic acid lateral flow immunoassay . It is a hand-held, portable device that can be programmed with a smartphone or laptop application, either through USB cable or Bluetooth connection. The latest model, mini16, has an affordable price of approximately 800 USD  and can process 16 samples per run. The mini16 can run on mains power, but also on a portable and solar-chargeable power pack, making the system completely autonomous and suitable for rural or emergency settings with unstable or no electricity supply. Finally, the result of the dbPCR is easily and rapidly read out with NALFIA, an immunochromatographic flow strip that can detect labelled PCR amplicons [Consequently, there is still a need for a highly sensitive, user-friendly and field-deployable diagnostic test for malaria that can discriminate mplicons –25. A NAP. falciparum infections of 1 parasite per microlitre (p/μL) of blood [Plasmodium and P. falciparum infections in blood.Earlier prototypes of the dbPCR-NALFIA assay have shown promising results in field evaluations, with sensitivity and specificity results up to 97.2% and 95.5%, respectively, using light microscopy as reference standard, and a detection limit for of blood , 23. In Plasmodium 18S rRNA gene: one that is highly conserved in the genus Plasmodium (the pan-Plasmodium target), and a second that is specific for P. falciparum [The dbPCR is a duplex reaction targeting two regions in the lciparum , 27. By lciparum . The dbPP. falciparum-infected EDTA blood sample and a Plasmodium-negative EDTA blood sample. As a first step, the samples were lysed at 98 °C for 10 min on the mini16 thermal cycler , a miniature endpoint PCR device  which can also be used for heat block protocols. The miniPCR smartphone application was used to programme the lysis protocol on the mini16 device through Bluetooth connection. After the lysis of the EDTA blood templates, 22.5 μL of the dbPCR reagent mix was added to each . The dbPCR was also run on the mini16 thermal cycler. Its protocol consisted of an initial activation step of 1 min at 98 °C, followed by 10 cycles of 5 s at 98 °C, 15 s at 61 °C and 30 s at 72 °C; next, 28 cycles of 5 s at 98 °C, 15 s at 58 °C and 30 s at 72 °C; and a final extension step of 72 °C for 2 min.The template format for the dbPCR is 2.5 μL of EDTA-anticoagulated blood. Every mini-dbPCR-NALFIA run includes controls, which are a Plasmodium amplicon was positive, it indicated the presence of Plasmodium infection. If the second anti-FITC test line for the fluorescein amidite (FAM)-labelled P. falciparum amplicon was also positive, the sample was infected specifically with P. falciparum . A sample with a positive pan-Plasmodium line and an absent P. falciparum line was classified positive for a non-falciparum malaria species, i.e. Plasmodium vivax, Plasmodium malariae, Plasmodium ovale or Plasmodium knowlesi. When only the P. falciparum line was visible, this result was interpreted to be positive for this species. A NALFIA test was considered invalid when the flow control line was absent.Read-out of the results was done with NALFIA . The test strip consists of a sample absorption pad, a conjugate pad with neutravidin-labelled carbon binding to the amplicons’ biotin label, and a nitrocellulose membrane coated with anti-digoxigenin (Dig) and anti-fluorescein isothiocyanate (FITC) antibody lines detecting and visualizing the amplicon-carbon complex. A third line on the membrane functions as a flow control Fig. . After cPlasmodium and P. falciparum targets was determined by testing 23 aliquots of a tenfold dilution series of a FCR3 ring-stage P. falciparum culture. The parasite density of the culture was determined by light microscopy. Dilutions were made in Plasmodium-negative EDTA blood from the Dutch blood bank. Tested parasite densities ranged from 1000 to 0.1 p/μL. LoD was defined as the lowest parasite density that was detected with 90% confidence (≥ 21 of 23 runs).The limit of detection (LoD) for the pan-P. falciparum and 0.1 p/µL for P. vivax [Plasmodium species had been determined with expert microscopy. This set included 23 P. falciparum, 3 P. vivax, 2 P. ovale and 1 P. malariae infections. The P. falciparum samples had been quantified microscopically and ranged from 106 to 102 p/μL; the parasite counts of the non-falciparum malaria samples had not been determined at the time of microscopic examination. The 58 Plasmodium-negative samples comprised 19 samples from the Dutch blood donors, 16 samples from intensive care unit patients and 23 samples from malaria-suspected returned travellers with a negative Alethia diagnosis. The operator that tested all samples with mini-dbPCR-NALFIA was blinded to the reference test outcomes.To determine the laboratory sensitivity and specificity of the mini-dbPCR-NALFIA, a set of 87 blood specimen was tested, including samples from returned Dutch travellers with suspected malaria infection, Dutch blood donors, and intensive care unit patients from the Academic Medical Centre . All samples were derived from a pre-established Biobank at the Laboratory for Experimental Parasitology at the Academic Medical Centre. Both blood donors and intensive care unit patients did not travel to malaria-endemic areas in the 6 months before blood collection. The malaria status of all samples had been determined previously using the Alethia Malaria assay , a highly sensitive LAMP-based method for diagnosing malaria in non-endemic settings with a detection limit of 2 p/µL for P. vivax , 29. ForP. falciparum culture and five Plasmodium-negative blood samples. For the accordance assessment, one operator tested three sets of aliquots with mini-dbPCR-NALFIA on three consecutive days, using the same equipment and dbPCR reagent batch numbers. To determine the concordance of the mini-dbPCR-NALFIA, five different operators from the same laboratory each tested a set of sample aliquots once. All five operators were blinded to the nature of the samples and used the same equipment and dbPCR reagent batch numbers.Accordance and concordance are measures to express, respectively, the repeatability (intra-operator variability) and reproducibility (inter-operator variability) of qualitative tests , 31. To Plasmodium target, the P. falciparum target and the overall assay. The Clopper-Pearson Exact method was used to calculate the 95% confidence interval (CI) of the sensitivity and specificity. Accordance and concordance were calculated in random framework, using the formulae proposed by Van der Voet and Van Raamsdonk (2004): 0 the proportion of negative results, p1 the proportion of pan-Plasmodium single positive results (i.e. only the pan line), p2 the proportion of P. falciparum single positive results  and p3 the proportion of double positive results , for a particular sample i. For the random concordance, the following formula was used: 0,i, p1,i, p2,i and p3,i represent the proportion of negative, pan single positive, P. falciparum single positive and double positive results for a particular operator i [Sensitivity and specificity were calculated for the pan-erator i . The 95%erator i , 32.P. falciparum culture dilution series testing are displayed in Table Plasmodium test line and 10 p/µL for the P. falciparum line.The results of the Plasmodium samples, 28 tested positive for the pan-Plasmodium line in the mini-dbPCR-NALFIA, while 1 P. vivax sample was false-negative for this line. All 23 P. falciparum samples also showed the P. falciparum test line. 57 Plasmodium-negative blood samples were negative for both test lines with mini-dbPCR-NALFIA; 1 sample from a Dutch blood donor was false-positive for the P. falciparum line. None of the test samples had an invalid NALFIA result.Of the 29 Plasmodium line. The sensitivity of the P. falciparum test line was calculated to be 100% , and its specificity 98.4% . When the results of the two NALFIA test lines were combined, there were three possible outcomes: a non-falciparum infection, a P. falciparum infection and Plasmodium-negative. This approach resulted in an overall sensitivity of 96.6%  and specificity of 98.3%  of the mini-dbPCR-NALFIA.This resulted in a sensitivity of 96.6%  and a specificity of 100%  for the pan-. The overall accordance of all tested samples in a random framework was 93.7% . An overview of the accordance test results for the mini-dbPCR-NALFIA is shown in Table 3Table This study demonstrates that the mini-dbPCR-NALFIA is a robust, highly sensitive and specific tool for molecular diagnosis of malaria. It has a simpler workflow than traditional NAATs and requires much less resources. By incorporating the mini16 as portable, battery-powered thermal cycler, the mini-dbPCR-NALFIA can be used even in remote healthcare settings without an extensive laboratory infrastructure or stable power supply.P. vivax sample gave a false-negative result. This may have been due to a low parasite density, which is common in P. vivax infections [Plasmodium-negative sample could have been the result of carry-over contamination from a Plasmodium-positive sample during the preparation of the dbPCR or NALFIA.With an excellent overall sensitivity of 96.6% and specificity of 98.3%, the diagnostic accuracy of the mini-dbPCR-NALFIA is similar to that of traditional molecular techniques for malaria diagnosis, such as conventional PCR, qPCR and nested PCR . One P. fections . UnfortuPlasmodium line and 10 p/μL for the P. falciparum line demonstrate the high sensitivity of the mini-dbPCR-NALFIA for low falciparum parasite densities. Although the LoD of extremely sensitive nested and qPCR techniques can go as low as 0.1 p/μL [falciparum parasitaemias than light microscopy and RDTs, which generally fail to detect infections below 50 to 200 p/µL [falciparum cases with sub-microscopic infections [falciparum samples were available for this study, additional evaluation of the LoD of the mini-dbPCR-NALFIA for these other Plasmodium species is warranted.The LoDs of 100 p/μL for the pan-0.1 p/μL –37, most200 p/µL . As such200 p/µL –41. On tfections . As no qP. falciparum blood dilution series and five malaria-negative blood samples, the mini-PCR-NALFIA showed a high accordance of 93.7%, demonstrating the robustness of the method. Discordant results were mainly observed for parasite densities < 10 p/μL, which are close to the LoD of the test. At such low Plasmodium DNA concentrations, stochastic variations tend to have a more prominent influence on the assay’s outcome. This phenomenon was also believed to be the main reason for the concordance being 84.6%. The laboratory experience of the different operators in the concordance evaluation ranged from basic to proficient. They were only given written and oral instructions, which was sufficient for them to correctly perform the mini-dbPCR-NALFIA. This observation underlined its simplicity and user-friendliness.When analysing a falciparum infections [Plasmodium species in one reaction. This issue is not encountered with mini-dbPCR-NALFIA, a duplex assay that can distinguish falciparum malaria from infections with other Plasmodium species. To further evaluate the performance of the mini-dbPCR-NALFIA for diagnosis of (submicroscopic) infections with P. vivax, P. malariae and P. ovale., additional research is required, since this study tested only a limited number of non-falciparum malaria blood samples.Compared to other molecular methods for malaria diagnosis, mini-dbPCR-NALFIA shares some characteristics with LAMP, which also has a simplified protocol with easy read-out and high accuracy for diagnosing malaria, including low density fections , 44. HowThe adaptation of the assay described by Roth et al.  to operafalciparum malaria species and identify mixed infections. Although the vast majority of malaria cases in Africa is caused by P. falciparum, the relative contribution of P. vivax, P. malariae and P. ovale infections in this region appears to be increasing [P. falciparum and a P. vivax test line. In the same way, the mini-dbPCR-NALFIA also has the potential to be modified to detect other blood-borne pathogens.A limitation of the current mini-dbPCR-NALFIA is its inability to differentiate between the non-creasing –48. FortP. falciparum HRP2 antigen persistence in the blood after clearance of the parasites [Plasmodium DNA in blood can remain positive up to seven weeks after curative malaria treatment [In areas with high malaria transmission, the mini-dbPCR-NALFIA could be a valuable alternative to RDTs, which are likely to suffer from false-positive results due to arasites , 49, 50.reatment , 52. ThiP. falciparum and non-falciparum infections is possible thanks to its duplex format. A handheld miniature thermal cycler makes the assay well-adapted to resource-poor conditions in malaria endemic regions. The high diagnostic accuracy and low LoD of the mini-dbPCR-NALFIA could make it a valuable tool in many malaria control programmes, especially for detection of asymptomatic and low-density cases in near-elimination areas. A phase-3 field trial is currently being conducted to evaluate the potential of the mini-dbPCR-NALFIA in different epidemiological settings.The mini-dbPCR-NALFIA is an easy-to-use method for sensitive and specific diagnosis of malaria. Compared to other simplified molecular diagnostics, it has the advantages that there is no need of prior sample processing and that differentiation of